LC-MS/MS measurements of ketoacids isotopologues distribution in HeLa cells after 1 day of intramitochondrial pyruvate influx by Grubraw

We analysed 13C metabolic inclusion into ketoacids in HeLa cell lines stably expressing Pseudomonas aeruginosa DadA (FAD-dependent D-amino acid dehydrogenase) gene with mitochondrial targeting (named Grubraw) after the addition of 12C D-alanine comparing to no D-alanine. We used HeLa with functional DadA and mutated DadA for control. In cells expressing functionally active DadA, D-alanine generates additional influx of intra-mitochondrial pyruvate. If cells grows on labelled carbone source and unlabeled D-alanine is used, this upcoming pyruvate shifts original mass-isotopologue distribution that can be registered by LC-MS. Our cells were cultured for in a medium with labeled glucose or glutamine. To obtain current data, we changed growth media to pure one, added D-alanine, and incubated cells under these conditions for 24 hours before extraction. Using this scheme, we focused on a long-time effects of Grubraw activation. We publish original RAW data from Orbitrap mass-spectrometer of intra- and extracellular ketoacid analysis. In “G” folders we put experiments with 1,2-[13]C-glucose and unlabelled glutamine. In “Q” folders, there are measurements with 5-[13]C-glutamine and unlabelled glucose. All files are named in a similar way. C12/C13 in the beginning indicates using of isotopically labeled carbon source or non-labeled one. “M”/“D” at the second position indicate using a cell line with mutated (M) or functional (D) DadA. “Plus”/“minus” indicates the addition of D-alanine. First number is a biological replicate, second number - technical replicate.本研究针对稳定表达铜绿假单胞菌（Pseudomonas aeruginosa）DadA基因（黄素腺嘌呤二核苷酸（FAD）依赖型D-氨基酸脱氢酶）且携带线粒体靶向序列的HeLa细胞系（命名为Grubraw），以未添加D-丙氨酸的组别为对照，分析了添加12C标记D-丙氨酸后，13C代谢物向酮酸中的掺入情况。本研究以携带功能性DadA与突变型DadA的HeLa细胞作为对照。在表达功能活性DadA的细胞中，D-丙氨酸可驱动线粒体内丙酮酸的额外流入。若细胞培养于同位素标记碳源的培养基中，同时添加未标记的D-丙氨酸，由此生成的丙酮酸会改变原本的质量同位素分布，该变化可通过液相色谱-质谱联用（LC-MS）进行检测。 我们的细胞培养于添加了同位素标记葡萄糖或谷氨酰胺的培养基中。为获取本实验数据，我们将细胞更换至基础培养基，添加D-丙氨酸后，在该条件下孵育细胞24小时再进行样本提取。通过该实验方案，我们聚焦于Grubraw激活的长期效应。 我们公开了源自轨道阱（Orbitrap）质谱仪的原始RAW数据，用于胞内与胞外酮酸的分析。在"G"文件夹中，我们存放了使用1,2-[13C]葡萄糖与未标记谷氨酰胺的实验组数据；在"Q"文件夹中，则存放了使用5-[13C]谷氨酰胺与未标记葡萄糖的实验组数据。所有文件的命名规则统一：文件名开头的"C12"/"C13"代表所使用的同位素标记碳源或非标记碳源；第二位的"M"/"D"分别对应使用携带突变型（M）或功能性（D）DadA的细胞系；"Plus"/"minus"表示是否添加了D-丙氨酸；第一个数字为生物学重复样本编号，第二个数字为技术重复样本编号。